Without limiting the scope of the invention, its background is described in connection with tissue engineering and angiogenesis as an example. Further, for purposes of the ensuing discussion, description and claims of the present invention, the terms “protein”, “polypeptide” and “peptide” may be used interchangeably, although it will be appreciated by those skilled in the art that biological distinctions may be drawn between them and, as such distinctions do not affect the operation of the present invention, such distinctions as may be drawn are contemplated by the scope of the present invention.
The advent of recombinant DNA and protein technology makes possible the production of significant quantities of both DNA and proteins for use in the clinical setting. The appeal of recombinant therapeutics is enhanced by delivery systems that provide controlled pharmacokinetics of the desired therapy. Paramount to the development of such a delivery system is the assurance that the biological activity of the material is preserved throughout manufacturing, storage, delivery, and release. Biodegradable microspheres have the potential to meet these requirements.
Methods are known to encapsulate various proteins within biodegradable microspheres. In particular, polypeptides have been incorporated into Poly(DL-lactide-co-glycolide) (PLGA) microspheres with varying degrees of success. PLGA is a polymer that has been used for many years as a biodegradable suture material. PLGA is biocompatible and degrades by hydrolytic cleavage into nontoxic molecules that are easily eliminated from the body (namely, lactic acid and glycolic acid). In addition to polypeptide microencapsulation, sustained delivery of polypeptides is also possible through the use of biodegradable microspheres. A non-exhaustive list of such polypeptides includes nerve growth factor, alpha, beta and gamma interferon, growth hormone, insulin erythropoietin, transforming growth factor beta, epidermal growth factor interleukin-2, basic fibroblast growth factor and vascular endothelial growth factor. PLGA has been described, therefore, as a desirable polymer for use as a drug delivery system. Preserving the biological activity of the microencapsulated polypeptide, however, has proven to be problematic and has retarded the development of microencapsulation drug delivery.
The double-emulsion technique is the most commonly reported method for manufacturing microspheres. According to this technique, protein dissolved in an aqueous solution is then emulsified in an organic solvent containing the dissolved PLGA. The aqueous-organic emulsion is then further emulsified in an aqueous alcohol phase to create an aqueous-organic-aqueous double emulsion. The alcohol phase extracts the organic solvent away from the PLGA in approximately one hour, leaving the protein entrapped in discrete droplets within solid microspheres. The process of emulsifying the aqueous protein solution in the organic solvent, however, can easily denature the protein.
Protein may be encapsulated into microspheres by known methods either in solution or as a solid. The incorporation of solid protein into a microsphere has previously been accomplished by the atomization-freeze (AF) process. The AF process requires the use of an ultrasonic atomizer with a custom designed spray nozzle. A description of the atomization-freeze technique is found in Putney S. D., Burke P. A., “Improving Protein Therapeutics with Sustained-release Formulations,” Nat. Biotechnol 1998; 16:153-157.
Another method for the encapsulation of solid proteins is described in Cao X., Schoichiet M. S., “Delivering Neuroactive Molecules from Biodegradable Microspheres for Application in Central Nervous System Disorders,” Biomaterials 1999; 20:329-339. Briefly, Cao and Schoichet dispersed ovalbumin powder in a solution of PLGA in chloroform using a Polytron homogenizer. The protein-polymer dispersion was added to an aqueous solution of 1% polyvinyl alcohol (PVA) and homogenized again to form an emulsion. The emulsion was added to more PVA solution and stirred continuous to evaporate the organic solvent. The microspheres were centrifuged, washed and freeze dried.
One problem with the method of Cao, et al., is the use of a single polymer species such as PLGA alone, or a PLGA/poly(eta-caprolactine) to fabricate the microspheres. Certain advantages in the release kinetics of the microspheres may be achieved by using a mixture of polymers rather than a single polymer species to fabricate the microspheres. Release kinetics are determined, in part, by the amount of bioactive substances loaded, the polymer or polymers used and the conditions of manufacture. The particle size of the microspheres is determined to a large extent by the manufacturing conditions such as polymer viscosity and the method of physical shearing used to produce the microspheres. Methods of shearing include but are not limited to homogenization with a tissue homogenizer or blender, ultrasound sonication, or vibrating with the use of a VORTEX® mixer. Smaller particles have a faster rate of degradation due to the increased ratio of surface area to volume. Thus, for microspheres composed of PLGA alone, the release of protein is generally regulated only by the physical erosion of the polymer, particularly where the protein/polymer ratio of the microsphere is low. Another problem with the Cao, et al., method is the use of a tissue homogenizer to form the polymer emulsion. In terms of commercial scale-up of microsphere production, a tissue homogenizer is impractical. Homogenizers have parts such as blades, rotors and containers that require cleaning and sterilization between each batch. The care and maintenance of homogenizers renders them problematic for the large scale production of pharmaceutical microspheres.